title: A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches.
abstract: BACKGROUND
  Recent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV.
  
  
  RESULTS
  We examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5' cyclization sequence (5' CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 10(1) TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use.
  
  
  CONCLUSIONS
  We have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, IN 46556, USA. jcarte11@nd.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Carter
authors.forename: Velmurugan
authors.initials: V
authors.lastname: Balaraman
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Kucharski
authors.forename: Tresa S
authors.initials: TS
authors.lastname: Fraser
authors.forename: Malcolm J
authors.initials: MJ
authors.lastname: Fraser
mesh_terms: D000330:Aedes; D000818:Animals; D002460:Cell Line; D021881:DNA, Catalytic; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D006046:Gold; D006801:Humans; D053758:Nanoparticles; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D021881:DNA, Catalytic; D012367:RNA, Viral; D006046:Gold